- Materials
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- Barcode Adapter (BCA)
- Consumables
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- 1.5 ml Eppendorf DNA LoBind tubes
- NEB Blunt/TA Ligase Master Mix (NEB, cat # M0367)
- Agencourt AMPure XP beads (Beckman Coulter, A63881)
- Nuclease-free water (e.g. ThermoFisher, AM9937)
- Freshly prepared 70% ethanol in nuclease-free water
- 10 mM Tris-HCl pH 8.5
- Equipment
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- Microfuge
- Hula mixer (gentle rotator mixer)
- Vortex mixer
- Magnetic rack, suitable for 1.5 ml Eppendorf tubes
- Ice bucket with ice
- Optional equipment
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- Agilent Bioanalyzer (or equivalent)
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Add the reagents in the order given below, mixing by pipetting between each sequential addition:
Between each addition, pipette mix 10-20 times.
Reagent Volume End prep DNA 30 µl Barcode Adapter 20 µl Blunt/TA Ligase Master Mix 50 µl Total 100 µl -
Mix gently by flicking the tube, and spin down.
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Incubate the reaction for 10 minutes at room temperature.
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Resuspend the AMPure XP beads by vortexing.
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Add 40 µl of resuspended AMPure XP beads to the reaction and mix by flicking the tube.
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Incubate on a Hula mixer (rotator mixer) for 5 minutes at room temperature.
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Prepare 500 μl of fresh 70% ethanol in nuclease-free water.
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Spin down the sample and pellet on a magnet. Keep the tube on the magnet, and pipette off the supernatant.
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Keep the tube on the magnet and wash the beads with 200 µl of freshly prepared 70% ethanol without disturbing the pellet. Remove the ethanol using a pipette and discard.
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Repeat the previous step.
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Spin down and place the tube back on the magnet. Pipette off any residual 70% ethanol. Briefly allow to dry.
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Remove the tube from the magnetic rack and resuspend pellet in 25 µl nuclease-free water. Incubate for 2 minutes at room temperature.
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Pellet the beads on a magnet until the eluate is clear and colourless.
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Remove and retain 15 µl of eluate into a clean 1.5 ml Eppendorf DNA LoBind tube.
- Remove and retain the eluate which contains the DNA library in a clean 1.5 ml Eppendorf DNA LoBind tube
- Dispose of the pelleted beads
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Quantify 1 µl of end-prepped DNA using a Qubit fluorometer.
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Dilute the library to a concentration of 10 ng/µl with nuclease-free water or 10 mM Tris-HCl pH 8.5.